Immuno-reactive peptide CTL epitopes of human cytomegalovirus

ABSTRACT

The invention provides a plurality of peptides (and immunologically functional variants thereof) which are immunogenic epitopes recognized by CD8 +  class I MHC restricted cytotoxic T-lymphocytes of patients harboring latent cytomegalovirus (HCMV) infection. The peptides are capable of activating CTLs and CTLp&#39;s in the absence of active viral replication, and thus are useful for eliciting a cellular immune response against HCMV by normal and immunodeficient subjects. Polypeptide and lipopeptide vaccines, with and without adjuvants, also are disclosed.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support in the form of grant no.CA30206 from the United States Department of Health and Human Services,National Cancer Institute. The government may have certain rights in theinvention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.08/950,064, filed Oct. 14, 1997 (abandoned), which is acontinuation-in-part of application Ser. No. 08/747,488, filed Nov. 12,1996 (abandoned).

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to human cytomegalovirus (HCMV), and inparticular to peptide fragments from a single subunit protein thatfunction as T-cell epitopes of HCMV in human beings. The peptidefragments are capable of directing human cytotoxic T lymphocytes (CTL)to recognize and lyse human cells infected with HCMV. The peptidefragments can independently direct HCMV-specified CTL to lyse cellsincubated with the peptide and which express HLA A, B or C genes.

2. Description of the Background Art

The HCMV genome is relatively large (about 235k base pairs) and has thecapacity to encode more than two hundred proteins. HCMV is composed of anuclear complex of nucleic acid (double-stranded DNA) surrounded bycapsid proteins having structural or enzymatic functions, and anexternal glycopeptide- and glycolipid-containing membrane envelope. HCMVis a member of the herpes virus family and has been associated with anumber of clinical syndromes.

HCMV infection is relatively common and is usually self-limiting in thehealthy, immunocompetent child or adult (L. Rasmussen, Curr. Top.Microbiol. Immunol. 154:221-254, 1990). Approximately ten percent (10%)of all newborn infants carry HCMV and the virus can cause severecongenital disease in the fetus or infant. Some of these newborn infantssuffer congenital birth defects. Other newborn infants carrycytomegalovirus for some time before they actually show symptoms of thedisease. For example, HCMV is a common cause of mental retardation inchildren who acquire the infection in utero from mothers carrying anactive infection.

Several studies have begun to question whether persistent and apparentlyasymptomatic HCMV infection in an otherwise healthy adult poses healthrisks in certain individuals. For example, individuals who haveundergone coronary angioplasty sometimes subsequently develop restenosisas a result of arterial remodeling. In one study, about one third ofsuch patients with restenosis had detectable HCMV DNA in their arteriallesions (E. Speir et al., Science 265:391-394 (1994)), whereas inanother study CMV seropositive patients were five times more likely todevelop restenosis than their seronegative counterparts (Y. F. Zhou etal., New England J. Med. 335:624-630 (1996)). These studies suggest thatdecreasing the number of HCMV infected host cells can benefit certainindividuals.

HCMV also has been associated with morbidity and mortality inimmuno-compromised patients. HCMV is an important consideration in thetreatment of patients suffering from Acquired Immunodeficiency Syndrome(AIDS). The defining complication is retinitis, which, if leftuntreated, can lead to blindness. Historically, CMV disease has been oneof the more devastating of the opportunistic infections (OI) that besetHIV-1-infected individuals whose CD4⁺ T cell level diminishes below100/mm³. Other disease manifestations of CMV viremia also appear as theCD4⁺ T cell counts drops below 100/mm³, including encephalitis,enteritis and pneumonia. At autopsy there is multi-organ involvement ofCMV disease in the preponderance of AIDS patients who had severe CMVretinitis.

Patients infected with HCMV often suffer impairment of some of theirvital organs, including the salivary glands, brain, kidney, liver andlungs, as a result of the effects of the disease. Furthermore, HCMV isassociated with a wide spectrum of classical syndromes includingmononucleosis and interstitial pneumonia. HCMV also has an oncogenicpotential and a possible association with certain types of malignanciesincluding Kaposi's sarcoma.

HCMV can cause opportunisitic infections resulting in a variety ofcomplications in, for example, immunosuppressed organ transplantpatients. Prior to the use of antiviral chemotherapy, HCMV infection hadbeen responsible for a substantial proportion of post-bone marrowtransplantation (BMT) complications (J. Meyers et al., J. Infect Dis.153:478-488 (1986)). The advent of drugs such as ganciclovir withsubstantial anti-CMV activity dramatically reduced complicationsassociated with post-BMT CMV infections (G. Schmidt et al. New EnglandJ. Med. 324:1005-1011 (1991) and J. M. Goodrich et al., New England J.Med. 325:1601-1607 (1991)). Ganciclovir is most effective whenadministered prophylactically before diagnosis of HCMV infection. Thisapproach has several negative aspects including a higher proportion ofrecipients becoming neutropenic (one third) and increased numbers ofconcomitant fatal bacterial and fungal diseases (J. M. Goodrich et al.,Ann. Intern. Med. 118:173-178 (1993)). An alternative approach in whichganciclovir was given when HCMV antigens or DNA are first detected byculture methods provided no survival advantage compared to prophylaxisor treatment post-disease for all patients (D. J. Winston et al., Ann.Intern. Med. 118:179-184 (1993)). Finally, because of the acute natureof the side-effects, there is a need for increased hospitalization andgrowth factor administration to treated patients which, coupled with thecost of ganciclovir prophylaxis, increases the cost of BMT after-care.

Because human cytomegalovirus is relatively common, yet is associatedwith extremely serious health conditions, a considerable effort has beenmade to study the biology of the virus with the aims of improvingdiagnosis of the disease as well as developing preventative andtherapeutic strategies.

The mounting of a CD8⁺ CTL response is believed to be an importantmammalian host response to certain acute viral infections. Theobservations that HCMV infection is widespread and persistent, and maybe reactivated and become clinically evident in the immunosuppressedpatient, have suggested that virus-specific T-cells, includingHCMV-specific CTL, play an important role in the control of persistentinfection and the recovery from CMV disease.

In humans, protection from the development of CMV disease inimmunosuppressed BMT recipients correlates with the recovery ofmeasurable CD8⁺ CMV-specified class I MHC-restricted T cell responses(Quinnan et al., N. Eng. J. Med. 307:7-13 (1982); Reusser et al., Blood78:1373-1380 (1991)). These observations led investigators to carry outclinical trials in which donor-derived CMV-specific CD8⁺ CTL wereinfused into BMT recipients as an alternative to ganciclovir prophylaxisand therapy (S. R. Riddell et al., Science 257:238-241 (1992)). Thetransfer of CD8⁺ CTL clones to allogeneic bone marrow transplantrecipients results in detectable CTL-based CMV immunity, andstatistically significant diminution of CMV disease after BMT (E. A.Walter et al., N. Eng. J. Med. 333:1038-1044 (1995)).

Although successful in application, this approach has the disadvantagethat it requires a sophisticated laboratory setup (which is also highlylabor-intensive and costly) to derive the HCMV-specific CTL in vitro tobe reinfused into a patient. A desirable alternative would be to delivera vaccine derived from HCMV that would impart immunity to a BMTrecipient, a solid organ recipient, a heart patient, an AIDS patient ora woman of child-bearing years, without the need for ex vivo expansionof HCMV-specific CTL. To develop such a vaccine, the viral proteinswhich cause the host to recognize HCMV in a protective manner must beidentified, so that their amino acid sequence information can bedetermined. No such vaccine presently is available, however.

The viral life cycle provides insight as to the most effective timeframe for targeting a vaccine to maximally disrupt virus production andspread. Following HCMV entry into the host cell and uncoating, the viralgenome is expressed sequentially via immediate early (0-2 hour), early(2-24 hour) and late (>24 hour) viral proteins. However, certain viralstructural proteins such as pp65 are chaperoned into the cell because oftheir existence in large quantity in the viral particle. Much attentionhas focused upon structural virion proteins as potential immunodominanttarget antigens for HCMV-specific CTL responses.

One viral structural protein, pp65, has been identified as a targetantigen for CMV-specific class I MHC restricted CTL derived from theperipheral blood of most asymptomatic CMV seropositive individuals (E.Mclaughlin-Taylor et al., J. Med. Virol. 43:103-110 (1994)).Importantly, CD8⁺ class I MHC restricted CTL specific for pp65 willrecognize autologous HCMV-infected cells without the requirement forviral gene expression, presumably as a result of processing of theinternal depot of pp65 that is transferred into the cell duringinfection (M. J. Gilbert et al., J. Virology 67:3461-3469 (1993)). CTLagainst pp65 or pp150 (another matrix protein that is recognizedfrequently) are able to recognize and lyse HCMV-infected cells in vitrowithin an hour of infection in the absence of viral gene expression (S.R. Riddell and P. D. Greenberg, Curr. Top. Microbiol. Immunol. 189:9-34(1994)). Thus, these CTL may represent an important effector cell forlimiting HCMV reactivation and progression to CMV disease, and such acellular immune response in both immunocompromised and normalindividuals would be extremely important (C.-R. Li et al., Blood83:1971-1979 (1994)). Alternatively, CTL recognizing envelope proteinsare not a substitute for pp65 and pp150 CTL because they are rarelyfound, arising late in infection and they are poor lytic effectorsbecause of the down-regulation of the required Class I MHC molecules (M.J. Gilbert et al., J. Virology 67:3461-3469 (1993)). Finally, the HCMVmajor protein IE, produced abundantly early after infection, isspecifically inhibited from being a stimulator of CD8⁺ CTL by aCMV-dependent blockade of its presentation (M. J. Gilbert et al., Nature[London] 383:720-722, 1996). Therefore, vaccines stimulating immunityagainst pp65 or pp150 would be the preferred mechanism for elicitingprotective immunity against CMV infection.

It has been established that individual MHC Class I moleculespreferentially bind peptides of a given motif and that the amino acidsequence of specific positions of the motif are invariant, allowing agiven peptide to bind to MHC Class I molecules with high affinity. Theseare referred to as "anchor positions" (K. Falk et al., Nature351:290-296 (1991)). Later studies have suggested that amino acidpositions other than the anchor positions also contribute to thespecificity of peptide binding to MHC Class I molecules. Additionally,residues at positions within the CTL epitope which do not interact withMHC may interact with T cells, presumably by binding the T Cell receptor(TCR). The binding of peptide amino acid residues to MHC or TCRstructures is independently governed, so that substitution of TCRbinding amino acid residues in many cases will not interfere withbinding to the MHC molecule on the surface of an antigen presentingcell.

Edman degradation followed by N-terminal sequence analysis has been usedto sequence the peptide mixture which is bound to the MHC class Ipeptide binding groove. In most cases the length of these peptides isbetween 9 and 11 amino acids. Mass spectrometry of HPLC separatedpeptide mixtures can elucidate the primary sequence of individualpeptides. Peptide fragments which bind to MHC identified in this mannerare referred to as "naturally processed epitopes." Alternatively, onecan predict which peptides of a given length, between 9-11 amino acids,will optimally bind to individual HLA Class I alleles based on theirconformity to a motif (K. Falk et al., Nature 351:290-296 (1991)). Onesuch motif has been established for HLA A*0201. Positions 2 and 9 of anonapeptide are anchor residues for HLA A*0201, with minor contributionsto binding from positions 1, 4, 3, 5, 6, 7, 8 in decreasing order ofimportance to binding strength (J. W. Drijfhout et al., Human Immunology43:1-12 (1995)). Similar motifs have been established for decamers andundecamers for HLA A*0201. Correspondingly, unique amino acid motifshave been established for a subset of other HLA A and B alleles topredict binding peptides between 8-11 amino acids (H. G. Rammensee etal., Immunogenetics 41 (4):178-228 (1995)).

It is recognized that CTL are an important mechanism by which amammalian organism defends itself against infection by viruses andpossibly cancer. A processed form of, e.g., a viral protein minimalcytotoxic epitope (MCE) in combination with MHC Class I molecules isrecognized by T cells, such as CD8⁺ CTL. Functional studies of viral andtumor-specific T cells have confirmed that an MCE of 8-12 amino acidscan prime an antigen presenting cell (APC) to be lysed by CD8⁺ CTL, aslong as the APC expresses on the cell surface the correct MHC moleculethat will bind the peptide.

It has been shown that the route of entry of a protein into the celldetermines whether it will be processed as an antigen bound to eitherMHC Class I or II molecules. The endogenous or Class I pathway ofprotein degradation is often used by infectious viruses when they arepresent within cells. Viral nucleoproteins which may never reach thecell surface as full length molecules are still processed within thecell, and degraded portions are transported to the surface via MHC ClassI molecules. Viral envelope glycoproteins, merely because they are cellsurface molecules, do not obligatorily induce CTL recognition. Rather,it has been found that viral nucleoproteins, predominantly in the formof processed epitopes are the target antigens recognized by CD8⁺ CTL (A.Townsend et al., Philos. Trans. R. Soc. Lond. (Biol). 323:527-533(1989)).

As it has become apparent that antigens entering the cell throughexogenous pathways (pinocytosis, etc.) are not typically processed andpresented by Class I MHC molecules, methods to introduce proteinsdirectly into the cytoplasm have become the focus of vaccine developers.An approach that had gained favor was to use recombinant vacciniaviruses to infect cells, delivering a large amount of intracellularantigen. The enthusiasm for using vaccinia viruses as vaccines hasdiminished, however, because these viruses have the potential to causedisease in immunosuppressed people, such as BMT recipients. Anotherapproach to vaccination is to mix an antigenic protein with an adjuvantand introduce the mixture under the skin by subcutaneous injection.

Yet another potential approach to immunization to elicit CTL is to usethe MCE defined for a viral antigen in the context of a particular MHCrestriction element to boost a CTL memory response to a virus. Theability of an MCE to provide protective immunity to challenge by alethal dose of an infectious virus has been discussed in the literature.Vaccine developers have developed increasing interest in utilizing theMCE as the vaccine because it is capable of binding to MHC Class Imolecules through external binding of the cell surface molecules withoutthe need for internalization or processing. The MCE has been mosteffective as an immunogen when synthesized as a lipidated peptidetogether with a helper CD4 epitope (A Vitiello et al., J. Clin. Invest.95:341-349 (1995) and B. Livingston et al., J. Immunol. 159:1383-1392,1997). Other modifications of the bivalent vaccine include inclusion ofa signal sequence (KDEL) for endoplasmic reticulum retention andtargeting to attain maximum activity. There is also evidence in theliterature that an MCE presented by particular types of APC (e.g.dendritic cells) may cause a primary immune response to occur in theabsence of viral infection or prior contact with the virus or tumorcell.

Accordingly, in spite of significant efforts towards identifying theHCMV proteins that are recognized by CTLs, as well as the specificidentification of the HCMV late structural protein pp65, improvedmethods of preventing and treating HCMV infection are needed.Introduction of CMV-specific CTL into a recipient is not a universallyapplicable and practical strategy to confer immunity to all thoseat-risk individuals who may need to be immunized against HCMV infection.

SUMMARY OF THE INVENTION

Accordingly, one aspect of the present invention relates toimmunologically active peptides, and functional variants thereof,capable of eliciting a cellular immune response to HCMV in humans. Thepeptides are capable of directing human CTL to recognize and lyse humancells infected with HCMV. Such immunologically active peptides, inassociation with an MHC Class I molecule, are recognized by CTLs ofindividuals having a latent (inactive) HCMV infection.

Another aspect of the present invention provides a method of augmentingthe immune system of a patient in need thereof (i.e., a patientharboring a latent or active CMV infection) by administering at leastone immunologically active peptide according to the present inventionthat will be recognized by CTLs and/or CTLps (CTL precursors) of thepatient.

In yet another aspect of the invention, at least one immunologicallyactive peptide is administered to uninfected individuals to provideimmunity against future infections by HCMV. Such a peptide may beadministered in the form of a peptide or lipopeptide vaccine, optionallywith an adjuvant.

Alternatively, the peptide(s), may be administered in the form of acellular vaccine via the administration of autologous or allogeneicantigen presenting cells or dendritic cells that have been treated invitro so as to present the peptide on their surface.

Yet another aspect of the invention is a method to augment the immuneresponse of an individual who is latently infected with CMV and is atrisk for reactivation of CMV infection, wherein T cells are removed froman individual and treated in vitro with a peptide of the presentinvention. The resulting CMV-reactive CTL are reinfused autologously tothe patient or allogeneically to, for example, a BMT recipient.

In yet another aspect, a method to confer immunity against an HCMVinfection to a previously uninfected individual includes the steps ofremoving T cells from the individual, exposing the T cells in vitro to apeptide of the present invention and then reinfusing the resultingHCMV-reactive CTL to the individual.

The peptides of the present invention also may be administered topreviously infected or uninfected patients, or in vitro to T cells, inthe form of a polynucleotide (DNA-based) vaccine, wherein a suitablegene transfer vector, such as a plasmid or an engineered viral vectorthat contains DNA encoding the peptide fragment under the control ofappropriate expression regulatory sequences, is administered to thepatient or to T cells in culture.

In yet another of its aspects, the present invention provides avaccinia, canarypox or other eukaryotic virus vector containing a DNAsequence encoding the immunologically active peptide fragment. Thevector infects an antigen presenting cell which in turn presents antigenthat will be recognized by CTLs of patients having a latent (inactive)HCMV infection.

An additional aspect of the invention relates to diagnostic reagents fordetection of the presence of active versus quiescent HCMV infections.The peptides according to the present invention can directly stimulateCTLp in vitro and therefore can be used in an assay to determine thedegree of immunostimulation being caused by HCMV. The peptides can alsobe used to distinguish individuals who are seropositive from those whohave not been exposed to HCMV (seronegative individuals). T cells from apatient can be contacted in vitro with APC that have been primed with apeptide according to the present invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the cytotoxic response elicited by peptides in the absenceof lipidation.

FIG. 2 shows the cytotoxic response elicited by monolipidated peptides.

FIG. 3 shows the cytotoxic response elicited by dilipidated peptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to one aspect of the present invention, a nonapeptide (9 aminoacid peptide) of the sequence NLVPMVATV (pp65₄₉₅₋₅₀₃) (SEQ ID NO:1) isan immunogenic epitope of pp65 from CMV laboratory strains AD169 andTowne and all wild type isolates examined to date which is recognized byCD8⁺ Class I MHC restricted cytotoxic T-lymphocytes of patientsharboring latent CMV infection. The peptide is capable of activatingCTLs in the absence of active viral replication, and thus is useful foraugmenting the immune system of normal and immunodeficient patients, aswell as in the study of the Class I antigen processing pathway for HCMVproteins. The nonapeptide has amino acid residues in positions 2 and 9which are the preferred residues at those positions for interaction withthe HLA A*0201 and certain subtypes of HLA A*02XX, where XX=subtypes02-22 (J. W. Drijfhout et al., Human Immunology 43:1-12 (1995)).Nonetheless, other less preferred amino acid residues may replace thepreferred anchors, whereupon the peptide can continue to exhibit thecapacity to bind HLA A*0201 and certain subtypes of HLA A*02XX with theability to stimulate HCMV-specific CD8⁺ CTL.

Thus, in one aspect, the present invention provides an immunologicallyactive peptide, capable of eliciting a cellular immune response to humancytomegalovirus infection, of the preferred sequence:

NLVPMVATV (SEQ ID NO:1).

Sequence variants of the preferred peptide include peptides of thesequence NX₁ VPMVATX₂ wherein X₁ is L,I,M,T or V, and X₂ is V,A,C,I,L orT (SEQ ID NO:2). The invention includes the construction and selectionof other functional sequence variants, which can be carried out by thoseskilled in the art based upon the present disclosure. The peptide or thestructural variants disclosed herein also can be a functional part of alonger peptide which produces the immunological effects disclosedherein.

Other immunologically active peptides according to the present inventioninclude the peptides:

YSEHPTFTSQY (SEQ ID NO:3)

which binds to HLA A*01XX including A*0101 and subtypes thereof.Sequence variants of this peptide include peptides of the sequenceYXEHPTFTSQY wherein X is S, T or L (SEQ ID NO:4). The invention includesthe construction and selection of other functional sequence variants,which can be carried out by those skilled in the art based upon thepresent disclosure. The peptide or the structural variants disclosedherein also can be a functional part of a longer peptide which producesthe immunological effects disclosed herein.

FVFPTKDVALR (SEQ ID NO:5)

which binds to HLA A*68XX including A*6801 and subtypes thereof.Sequence variants of this peptide include peptides of the sequence FX₁FPTKDVALX₂ wherein X₁ is V or T and X₂ is L, R or K (SEQ ID NO:6). Theinvention includes the construction and selection of other functionalsequence variants, which can be carried out by those skilled in the artbased upon the present disclosure. The peptide or the structuralvariants disclosed herein also can be a functional part of a longerpeptide which produces the immunological effects disclosed herein.

TPRVTGGGAM (SEQ ID NO:7)

which binds to HLA B*07XX including B*0701 and subtypes thereof.Sequence variants of this peptide include peptides of the sequenceTPRVTGGGAX wherein X is L, F, or M (SEQ ID NO:8). The invention includesthe construction and selection of other functional sequence variants,which can be carried out by those skilled in the art based upon thepresent disclosure. The peptide or the structural variants disclosedherein also can be a functional part of a longer peptide which producesthe immunological effects disclosed herein.

FPTKDVAL (SEQ ID NO:9)

which binds to HLA B*35XX including B*3502, B*3504, B*3506 and othersubtypes thereof with compatible peptide binding sites. The inventionincludes the construction and selection of other functional sequencevariants, which can be carried out by those skilled in the art basedupon the present disclosure. The peptide or the structural variantsdisclosed herein also can be a functional part of a longer peptide whichproduces the immunological effects disclosed herein.

EXAMPLE 1 Derivation of T-cell Clones

Methods for deriving T-cell clones from CMV seropositive individualshave been described in the literature (see above references). Forty tofifty milliliter samples of whole peripheral blood were obtained fromCMV seropositive volunteers (detected by standard antibody methods). Thewhite blood cells (WBCs) were separated using Ficoll-HyPaque (DuPont)density gradient centrifugation. The whole blood was first centrifugedfor 10 minutes at 1400 rpm in a tabletop centrifuge to reduce the numberof red blood cells. The buffy coat was diluted to 12 ml with phosphatebuffered saline (PBS), and 6 ml were layered on top of 1/2 volume ofFicoll-HyPaque. The top layer was removed after centrifugation at 2000rpm in a tabletop centrifuge for 15-30 minutes. The interface containingthe WBC was removed, diluted in PBS and recentrifuged for 8-12 minutesat 1000 rpm, which caused the WBC to pellet. The cells were againresuspended in PBS and washed as above one additional time. Four to fivemillion WBC/ml were resuspended in T cell medium (TCM) with human serumobtained from pooled AB+ (blood group) CMV seronegative donors (HAB).

EXAMPLE 2 Derivation of LCL Antigen-Presenting Cells

Simultaneously, an autologous antigen presenting cell line was preparedby Epstein Barr virus immortalization of PBL (see Current Protocols inImmunology, Unit 7.22, Wiley-Liss Press (1993)). Deriving the CTLs andantigen presenting cells from the same individual ensures HLA matchingbetween the cell lines.

EXAMPLE 3 In vitro Stimulation by HCMV

To initiate the in vitro stimulation of the WBC, a monolayer ofautologous dermal fibroblasts obtained from the same volunteers as theWBC was established by plating the cells in 12-well plates at 10⁵cells/ml/well in DMEM-10% HAB for 24 hours. After 24 hours in culturethe fibroblasts were infected with CMV virions (AD169 or Towne strain)for 2 hours at a multiplicity of infection of between 1 and 5. Themedium and virus were aspirated from the monolayer, and 1 ml of freshDMEM-HAB was added. The monolayer was incubated in the medium for anadditional 4 hours, following which time, the medium was aspirated. Twomilliliters of medium containing 8-10 million WBCs were added per wellcontaining CMV infected fibroblasts. The WBCs and fibroblasts werecultured in RPMI-1640 (Irvine Scientific) containing 50 U/ml penicillin,50 μg/ml streptomycin, 4 mM L-glutamine, 25 μM 2-mercaptoethanol, 10 mMHEPES and 10% HAB (TCM-HAB). This was termed the first stimulation, andthe cells were co-incubated for 7 days. TCM-HAB may be replaced if itbecomes spent, or the culture may be expanded if there is vigorous cellgrowth.

The WBCs were re-stimulated on day 7 by plating onto a fresh monolayerof CMV-infected autologous fibroblasts prepared as described above. Inaddition, γ-irradiated (2500 rad) autologous PBL (5-fold over WBC) wereadded as feeder cells, and the medium was supplemented with recombinantIL-2 (10 IU/ml, Chiron-Cetus) on days 2 and 4 of this secondstimulation. Wells which exhibit rapid cell growth require new mediumcontaining IL-2 as the medium becomes spent.

After 12-16 days in culture, the cells were harvested and assayed forrecognition of CMV matrix proteins in a chromium release assay (CRA).The CRA was performed by preparing APCs as target cells which areautologous or HLA-mismatched to the T-cell clone by infection withrecombinant vaccinia viruses containing the DNA for HCMV proteins suchas pp28 (pp28vac), pp65 (pp65vac) and pp150 (pp150vac) or wild-typevirus strain WR.

After overnight infection, the APCs were incubated with chromium-51, andthe assay was carried out as described (Current Protocols in Immunology,Wiley-Liss Press, Unit 7.17, (1993)). In the CRA, the vaccinia infectedAPC (target cells) were loaded with chromium-51 and then mixed withcells from the T-cell clone (effector cells). Preferably, the cells aremixed at a series of effector:target cell ratios varying, for examplefrom 20:1 to 1:1. After a 4 hour incubation period, the medium in whichthe cells were incubated was harvested. The release of radioactivityinto the medium (R_(e)) was quantitated with a gamma scintillationcounter. The extent to which infected antigen presenting cells exhibitspontaneous lysis and the release of radioactivity (R_(s)) in theabsence of CTLs was established for each virus vector. The maximumamount of radioactivity incorporated into and releasable by the targetcells (R_(max)) was established by lysis of target cells in a detergent(1% Triton X100; Sigma) solution. Percentage cytotoxicity can beexpressed as:

    100×((R.sub.e))-((R.sub.s))/((R.sub.max)-(R.sub.s)).

Assays were deemed unacceptable and were repeated unless spontaneousrelease (R_(s)) was less than 30%.

Analysis of the assay was as described, with a positive result indicatedby specific recognition of pp65vac infected autologous APC. A positiveresult for pp65 indicates that, in the tested polyclonal population,there are T cells which recognize the pp65 HCMV protein expressed by thevirus.

EXAMPLE 4 Procedure for Identification of the CTL Epitope

WBC stimulated two times by HCMV on dermal fibroblasts were cloned bylimiting dilution in 96 well U-bottom plates as follows. After 2 HCMVstimulations, the WBC were depleted of CD4⁺ T cells by incubation withparamagnetic beads conjugated to anti-CD4 antibodies by negativeselection. The resulting population was generally between 90-95% CD8⁺, areliable T cell subset marker, and generally 99% CD3⁺, a marker for mostperipheral blood T cells as assayed by either flow cytometry orfluorescence microscopy. This final population was plated at aconcentration between 0.3-3 cells per well in a final volume of 150 μl.Each well also contained γ-irradiated 1.0-3.0×10⁵ allogeneic peripheralblood mononuclear cells (PBMC) in TCM-HAB supplemented with 50-100 IU/mlrecombinant IL-2 (Chiron-Cetus) and 0.5 μg/ml PHA (Murex).

After 3 days of culture, the PHA was diluted 2-fold by exchanging 75 μlwith fresh culture medium supplemented with rIL-2. The wells weresupplemented with fresh rIL-2 every 3-4 days, and medium was replaced asnecessary. The cells were restimulated at between 12-14 days with freshallogeneic PBMC as described above, and the plates were carefullyobserved for growth in individual wells. Visible cell growth indicatesthe need to transfer the expanding T cells to larger wells. T cells wererestimulated every two weeks, and were transferred to progressivelylarger wells. At the stage of accumulation of several million cells,some were cryopreserved, and others were subjected to a further CRA. Inthis CRA, the targets were CMV infected fibroblasts, uninfectedfibroblasts, autologous LCL infected with wild type vaccinia or vacciniavirus expressing either pp28, pp65 or pp150. HLA mismatched fibroblastsand LCLs were used as controls. One T cell clone among several tested,which was designated 3-3F4, had the characteristics of being both CMVand pp65-specific, and was reactive only to autologous targets in aspecific manner. Other T cell clones with different HLA phenotypes wereinitially isolated in the same way, except that the initial peripheralblood sample came from different volunteers.

The HLA element which restricted the recognition of the T cell clone3-3F4 to pp65 was identified. A series of LCL were used as targets thatwere singly autologous with each HLA allele of the 3-3F4 cell line. Eachtarget was separately infected with pp65vac, wild type vaccinia, or notinfected at all. The results showed that only the LCL that wereautologous to the HLA A*0201 allele were being recognized and killed bythe 3-3F4 T cell line. It was also established that 3-3F4 is of the CD8⁺T cell subset, characteristic of CTL which recognize Class I restrictedpeptides. Whether the cell line was monoclonal was tested by carryingout PCR repertoire analysis using a series of 26 human Vβ gene segmentprimers. Only one of 26 primers gave a significant signal, the Vβ13.1primer, thereby demonstrating the apparent monoclonality of the 3-3F4 Tcell clone.

To identify the precise epitope or peptide recognized by the T cellclone 3-3F4, a series of vaccinia truncations that deleted the pp65protein from the carboxyl towards the amino terminus was used. A CRAutilizing autologous and HLA mismatched LCL as targets, infected withvaccinia viruses expressing truncation products of the pp65 protein, wasconducted. This experiment localized a region between amino acids 377and 561 that was necessary for killing of targets. Utilizing a smallsubset of amino terminal deletions, the region necessary for killing wasfurther localized between amino acids 477 and 561. utilizing an indirectkilling assay, a monkey kidney cell line was transfected with themolecular HLA allele HLA A*0201 and portions of the pp65 gene tolocalize the region to a fragment containing amino acids 452-561. Thefinal determination of the peptide sequence that corresponds to thesequence bound by HLA A*0201, and is recognized by T cell clone 3-3F4was done by producing 9-10 amino acid peptides on the Synergy (AppliedBiosystems Model 432) peptide synthesis machine. Using publishedinformation, a series of candidate sequences were determined that hadsignificant characteristics of an HLA A*0201 binding sequence, and werelocated in the region between amino acid 452-561. These are:

                  TABLE 1                                                         ______________________________________                                        Amino Acid Sequence of                                                                         Position                                                     Peptide          Number    Motif   Score*                                     ______________________________________                                        ILARNLVPMV (SEQ ID NO:10)                                                                      491       A*0201  67                                         ELEGVWQPA (SEQ ID NO:11)                                                                       526       A*0201  59                                         RIFAELEGV (SEQ ID NO:12)                                                                       522       A*0201  55                                         NLVPMVATV (SEQ ID NO:1)                                                                        495       A*0201  63                                         ______________________________________                                         *(adapted from J. D'Amaro et al., "A computer program for predicting          possible cytotoxic epitopes based on HLA Class I peptide binding motifs,"     Human Immunology 43: 13-18 (1995)).                                      

Only one of these peptides (referred to as "the 495 peptide" or as"pp65₄₉₅₋₅₀₃ ") (SEQ ID NO:1) proved capable of priming the autologousLCL to be recognized and killed by the CD8⁺ CTL 3-3F4. Other 9-10 aminoacid peptides from pp65 that also followed the HLA A*0201 motif weretested in the CRA. None showed any activity. All peptides were examinedby HPLC on a Vydac C₁₈ column using acetonitrile/TFA as the movingphase. They were 70-80% pure or greater on average, and were used asdilutions from 0.1% acetic acid solution.

EXAMPLE 5

Use of the 495 Peptide to Induce Cell Lysis

Serial dilutions of the 495 peptide showed no change in activity between10 μM and 0.01 μM in priming the autologous LCL for killing by T cellclone 3-3F4 in a CRA. Half-maximal lysis occurred at close to 0.5 nMpeptide. The peptide-transporter deficient cell line T2 (D. B.Crumpacker et al., J. Immunol. 148:3004-3011 (1992)), which is HLAA*0201 positive, also was used to test the lower limits of sensitivityof the 3-3F4 T cell clone for recognition of the peptide in a CRA. Itwas found that as little as 0.1 nM peptide caused maximal lysis of T2cells, equivalent to the condition with 10 nM peptide. These experimentsdemonstrate that this minimal cytotoxic epitope is a strong binder tothe HLA A*0201 allele.

The 495 peptide depicted in Table 1 was prepared on a Synergy (AppliedBiosystems Model 432) peptide synthesis machine. Dermal fibroblasts wereprimed with the peptide, then loaded with chromium-51 via the followingprocedure. Dermal fibroblasts were incubated with 10 μM of the 495peptide for 2 hours at 37° C., and for the final hour with chromium-51.The peptide and chromium-51 were washed out of the medium.

T cell clone 3-3F4 (CD8⁺ CTL) derived from an HCMV seropositiveindividual (HLA A*0201 positive) as described above as capable ofrecognizing the HCMV-infected fibroblasts as well as the peptide-primedfibroblasts in a CRA. Uninfected and unprimed fibroblasts were notrecognized or killed by the T cell clone. In addition, an HLA-mismatchedfibroblast line without the HLA A*0201 allele found on the donorfibroblasts was not recognized or killed by the T cell clone when it waseither infected by HCMV or primed with the 495 peptide. Thus, the 495peptide of the present invention can serve as a "substitute" for thewhole HCMV virus in causing normal T cells to recognize and killfibroblasts as effectively or better than if they were infected withHCMV.

EXAMPLE 6

Generation of TNF-α by T Cell Clones

TNF-α is a T cell lymphokine that is cytotoxic for many cell types andmay contribute to the in vivo immune response mounted against an HCMVinfection. Cells of the 3-3F4 T cell clone were incubated withautologous fibroblasts pre-incubated with either whole HCMV virions orprimed with the 495 peptide of Table 1. Twenty four hours later,supernatants from the co-incubated cells were applied to an indicatorcell line in a bioassay as described above. The indicator cell line, aWEHI derivative, is sensitive to the cytotoxic action of TNF-α down tothe picomolar level.

Peptide-primed fibroblasts induced the production of as great or greaterlevels of TNF-α from T cell clone 3-3F4 as were the HCMV-infectedfibroblasts. The inventive 495 peptide did not induce TNF-α productionby either autologous fibroblasts incubated without T cells ornon-HLA-A*0201 expressing fibroblasts incubated with the T cell clone.

EXAMPLE 7

495 Peptide can Induce CTL from PBL of HCMV Seropositive Individuals

PBL from HCMV-seropositive individuals were plated onto 495peptide-primed autologous fibroblasts for 7 days. The once-stimulatedPBL were re-stimulated in a similar manner, either with or withoutdepletion of CD4 T cells. After two weeks, a CRA was performed using astargets either peptide-primed, CMV-infected, or untreated autologousfibroblasts. CD8⁺ T cells lysed significant percentages of the peptideprimed and CMV infected fibroblasts, but not untreated cells. Noautologous fibroblast targets were lysed by CD4-depleted PBL fromfreshly drawn blood under the same conditions.

EXAMPLE 8

Human Cell Lines Which Express HLA A2, With Molecular Subtypes OtherThan A*0201, are Sensitized to Lysis by the 495 Peptide

Twelve cell lines were subjected to CRA in which they were pulsed withthe 495 peptide (pp65₄₉₅₋₅₀₃) nonamer, loaded with chromium-51, andincubated with two different HLA A*0201 restricted CTL which recognizethe 495 peptide and HCMV. The specific cytotoxicity was calculated andis shown in tabular form below for the 1 μM and 1 nM concentrations ofthe 495 peptide. A plus sign (+) represents cytotoxicity greater than30%, a plus/minus sign (+/-) represents cytotoxicity between 15% and30%, and a minus sign (-) represents cytotoxicity less than 15%.

                  TABLE 2                                                         ______________________________________                                                    CTL 3-3F4      CTL VB-57                                          HLA A2 Subtype                                                                              1 μM                                                                              1 nM      1 μM                                                                            1 nM                                      ______________________________________                                        A*0201        +      +         +    +                                         A*0202        +      -         +    -                                         A*0203        +      -         -    -                                         A*0204        +      +         +    +                                         A*0205        +      -         +    +                                         A*0206        +      +         +    +                                         A*0207        +      +         +    +                                         A*0209        +      -         +    -                                         A*0210        +      +         +    +                                         A*0211        +      -         +    +/-                                       A*0217        +      -         +    +                                         ______________________________________                                    

The data show that all tested subtypes functionally bind the 495 peptideat 1 μM, and the cell lines are lysed by the HCMV and pp65 specific CTL.This data shows that the cell line subtypes shown here are capable ofbeing sensitized for lysis by the 495 peptide. The data also indicatethat cells from individuals who carry these subtypes can be sensitizedby the peptide for CTL recognition and lysis, albeit at a higherconcentration in most cases than what was found for HLA A*0201. Thus,individuals who carry any of these HLA A2 subtypes may respond to the495 peptide as a vaccine.

EXAMPLE 9

Animal Studies Using the 495 Peptide (SEQ ID NO:1)

A transgenic mouse model, the HLA A2.1 mouse (E. J. Bernhard et al., J.Exp. Med. 168:1157-1162 (1988)), was employed to test whether the 495peptide could stimulate CTLp lacking prior virus exposure and functionas a vaccine. Three mice were immunized subcutaneously at the base ofthe tail with 50 μg of the 495 peptide (pp65₄₉₅₋₅₀₃) or peptidep53₁₄₉₋₁₅₇ from p53 (M. Theobald et al., Proc. Natl. Acad. Sci. U.S.A.92:11993-11997 (1995)) together with 20 μg of the polyclonal helper Tlymphocyte (HTL) peptide (PADRE) (J. Alexander et al., Immunity.1:751-761 (1994)) emulsified in IFA (Incomplete Freund's Adjuvant).After twelve days, spleens were removed from immunized mice, a splenicsuspension was made, and the effector cells were restimulated for oneweek using p53₁₄₉₋₁₅₇ or pp65₄₉₅₋₅₀₃ peptide sensitized syngeneiclipopolysaccharide-treated splenic blast cells (P. A. Wentworth et al.,Eur. J. Immunol. 26:97-101 (1996)). Thereafter, for subsequent in vitrostimulations, the stimulator cells were Jurkat A2.1 cells, prepared byacid treatment and subsequent loading of peptides (Z. Tu et al., Journalof Surgical Research 69:337-343 (1997)). After two in vitrorestimulation cycles, the murine splenic effector population was testedfor recognition of p53₁₄₉₋₁₅₇ or pp65₄₉₅₋₅₀₃ peptide loaded T2 cells.There was substantial recognition of pp65₄₉₅₋₅₀₃ or p53₁₄₉₋₁₅₇ peptidein mice that had been appropriately immunized, without recognition ofthe non-immunizing peptide.

It was also demonstrated that murine splenic effectors recognizeendogenously processed pp65 in a CRA with human HLA A*0201 EBVLCLtargets infected with pp65vac (D. J. Diamond et al., Blood 90(5):1751-1767 (1997)). Further proof that the pp65₄₉₅₋₅₀₃ peptide causesrecognition of virus-infected cells came from a CRA using HCMV-infectedhuman fibroblasts as targets, and murine CTL derived from thepp65₄₉₅₋₅₀₃ peptide stimulation as the effector population. HLA A*0201fibroblasts infected with HCMV were capable of lysis by the CTL, whereasboth uninfected and mismatched fibroblasts were not recognized (D. J.Diamon et al. Blood 90 (5):1751-1767 (1997)). Taken together, theseresults clearly show that the splenic effector population from a primaryimmunization with pp65₄₉₅₋₅₀₃ are recognizing endogenously processedpp65 and HCMV in an HLA A*0201 restricted manner.

An additional study demonstrated that the 495 peptide can induce along-lived immune response in animals. Twelve mice were simultaneouslyimmunized with the 495 peptide+PADRE+IFA, and two additional mice wereimmunized with HTL+IFA along. AT two weeks, 6 weeks, 10 weeks and 14weeks after the immunization, the spleens from two immunized mice wereanalyzed for immunity against the 495 peptide or a control peptide fromhuman p53 (p53₁₄₉₋₁₅₇). In addition, at two and six weeks, miceimmunized without the 495 peptide were also sacrificed and their spleencells analyzed for immune responses against the 495 peptide or the humanp53 peptide. Percent specific cytotoxicity (cytotoxicity of 495 peptidetargets--cytotoxicity of p53 targets) was still at a level of 40% after14 weeks, whereas naive animals did not show any specific cytotoxicityabove 5%. This compared well with recent results from immunizing humanvolunteers with the Theradigm™ lipopeptide (B. Livingston et al., J.Immunol. 159:1383-1392 (1997)) in which there was an average maintenanceof 60% of the initial response 38 weeks after the final of fourimmunizations over an eighteen week period.

EXAMPLE 10

Lipidated Peptides Incorporating pp65₄₉₅₋₅₀₃ (SEQ ID NO:1) asIFA-Adjuvant Independent Vaccines in Animals

Although the use of adjuvants to enhance immunogenicity is a commonstrategy in animal studies, there are important limitations concerningtheir use in humans. To avoid having to use adjuvants, peptides whichincorporate lipid molecules were prepared. This strategy has beenefficacious in both animal (K. Deres et al., Nature 342:561-564 (1989))and human clinical studies (A. Vitiello et al., J. Clin. Invest.95:341-349 (1995)).

Either one or two palmitic acid moieties were attached to the aminoterminus of the 495 peptide, and a series of immunization studies wasconducted in the transgenic HLA A2.1 mouse. The primary structure of thedirectly lipidated peptides is shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Primary Structure of Peptides Used to Immunize                                HLA A2.1 Transgenic Mice                                                                 Adaptor    Epitope Seqence                                                                           Carboxyl                                    Lipid Molecule(s)                                                                        Sequence   (SEQ ID NO:1)                                                                             Terminus                                    ______________________________________                                        KSS-                                                                          NLVPMVATV-                        OH                                          1 PALMITIC ACID                                                               KSS-                                                                          NLVPMVATV- OH                                                                 2 PALMITIC ACID                                                               KSS-                                                                          NLVPMVATV- OH                                                                 ______________________________________                                    

Table 3. Peptides were synthesized on the ABI 432 (Applied Biosystems),and purified and analyzed as described (D. J. Diamond et al., Blood 90(5):1751-1767 (1997)). Palmitic acid (Aldrich) was dissolved indimethylformamide, and automatically coupled to the amino terminallysine. The amino terminal lysine of the monolipidated form of thepeptide was protected by two protecting groups, Fmoc and Boc. Only theFmoc group is cleaved during synthesis to allow for a single lipidmoiety to be added. For the dilipidated form, the amino terminal lysinewas protected with two Fmoc groups, therefore allowing two lipidmoieties to be added. Peptides also may be lipidated at other locationsby method well known in the art.

Separate mice were immunized with unmodified pp65₄₉₅₋₅₀₃, and either themonolipidated or dilipidated forms of the peptide together with thePADRE epitope. Because it has been previously shown that lipidatedpeptides will stimulate immunity without co-administration of Freund'sadjuvant (H. Schild et al., Eur. J. Immunol. 21:2649-2654 (1991)),immunization of mice with and without emulsifying the peptides in IFAwas compared. In a procedure similar to the procedure described inExample 9 above, twelve days post inoculation the mice weresplenectomized and the spleen cell pollution was restimulated twice invitro. The splenic effectors were then tested in a CRA withpeptide-loaded T2 cells, EBVLCL targets infected with pp65Vac, andHCMV-infected fibroblasts (D. J. Diamond et al., Blood 90 (5):1751-1767(1997)). The results demonstrate that the lipidated form of the 495peptide, without emulsification in adjuvant, induces an immune responsecapable of recognizing endogenously-processed pp65 and HCMV-infectedhuman cells. The monolipidated form appeared to induce a weaker immuneresponse (compare with and without IFA), whereas the unmodified freepeptide had no activity unless emulsified in IFA. (See FIGS. 1-3)

These data illustrate an advantageous immunization/vaccination procedureor use in warm blooded animals including humans. The procedure entailsthe mixture of two peptides in an aqueous solvent system (the lipidated495 peptide plus either PADRE or the 830-843 amino acid tetanus peptide,Table 3). The mixture is administered subcutaneously or via anotheracceptable and efficacious route to either HCMV seropositive orseronegative subjects. An additional set of booster injections may beemployed to enhance the immunity induced by primary vaccination.

EXAMPLE 11

Formulation of Lipidated Vaccines

The 495 peptide and functional sequence variants thereof can beformulated as a vaccine as a lipidated peptide with a covalent HTLepitope. The HTL epitope can be any peptide that has broad reactivity tohuman MHC class II to stimulate a classic helper response. Suchmolecules include but are not limited to amino acids 830-843 fromtetanus toxin (P. Panina-Bordignon et al., Eur. J. Immun. 19:2237-2242(1989)), HTL epitopes from HIV envelope protein (J. A. Berzofshy it al.,J. Clin. Invest. 88:876-884 (1991)), or a synthetic version (PADRE)predicted from known anchor residues (J. Alexander et al., Immunity1:751-761 (1994)).

The lipidation of the HTL+CTL epitope preferably is performed on theamino terminus of the HTL epitope, with the HTL epitope being aminoterminal to the CTL epitope. Suitable lipid moieties are known anddescribed in the literature. (H. Schild et al., Eur. J. Immunol.21:2649-2654 (1991); A. Vitiello et al., J. Clin. Invest. 95:341-349(1995); K. Deres et al., Nature 342:561-564 (1989)). Alternatively, theCTL epitope can be lipidated at its amino terminus, followed by the HTLepitope, or the lipid can be found at the carboxyl terminus followed byeither the CTL or HTL epitope(s). A three amino acid spacer can beinserted between the HTL and CTL epitope, or the epitopes can be fuseddirectly in frame. Alternatively the CTL epitope lipidated on its aminoterminus can be administered together with the HTL epitope, withoutcovalent attachment. The vaccine epitopes, regardless of primarystructure, may be injected s.c. into the forearm in a standardformulation buffer (PBS/10% DMSO or higher concentration/0.01%triflouroacetic acid or other acid or alcohol of the same or differentconcentration) once. Three to six weeks later, a booster injection ofthe same material may be administered. Multiple booster injectionsspaced three to six weeks apart can be subsequently administered, ifnecessary.

This injection series can be administered to a patient or at-riskindividual, or to the donor of a bone marrow transplant, who is eitherpositive or negative for the virus. Illustrative examples of lipidatedvaccine peptides include:

                                      TABLE 4                                     __________________________________________________________________________    N-terminal                                         C-terminal                     (Pam).sub.2-KSS QYIKANSKFIGITEAAANLVPMVATV                                                         (SEQ ID NO: 13)                                          (Pam).sub.3-CSS QYIKANSKFIGITEAAANLVPMVATV                                                         (SEQ ID NO: 14)                                          (Pam).sub.2-KSS AKXVAAWTLKAAAGGGNLVPMVATV                                                          (SEQ ID NO: 15)                                          (Pam).sub.3-CSS AKXVAAWTLKAAAGGGNLVPMVATV                                                          (SEQ ID NO: 16)                                      __________________________________________________________________________

wherein X is cyclohexylalanine or phenylalanine. As is the casethroughout, all amino acids are represented by their universalone-letter code. "Pam" is palmitic acid. The three-A or -G spacer(underlined) can be interchanged among vaccine peptides. The format ofthe peptides shown above can be described (from the amino terminus) as:lipid-K/CCS--HTL epitope (italics)--amino acid spacer (underlined)--CTLepitope. The positions of the CTL and HTL epitopes may be interchanged.The CTL epitope (or a functional sequence variant thereof) may befurther modified by adding a leader sequence and/or the amino acids KDELcan be appended to the carboxyl terminus for retention and targetinginto the endoplasmic reticulum. Palmitic acid or any suitable lipid maybe used, including but not limited to stearic acid, myristic acid,lauric acid, capric acid and decanoic acid.

EXAMPLE 12

Use of Combined HTL and CTL Epitope, Lipidated at the Amino Terminus, asa Single Component Vaccine in Transgenic Mouse Studies

Two further molecules, each containing lipid, HTL and CTL epitopes, weeconstructed and tested in mice:

                                      TABLE 5                                     __________________________________________________________________________    N-terminal                                      C-terminal                    A. (Pam)2-KSSAKXVAAWTLKAAANLVPMVATV                                                                     (SEQ ID NO:17)                                      B.  (Pam)2-KSSISQAVHAAHAEINEAAANLVPMVATV                                                                (SEQ ID NO:18)                                      __________________________________________________________________________

Nomenclature for Table 5 can be found in the legend to Table 4.

Molecule A (SEQ ID NO:17) is a vaccine that has the capability ofworking in mice and humans, whereas molecule B (SEQ ID NO:18) shouldonly be functional in mice. One hundred nanomoles of each of thesevaccine peptides in 25% dimethylsulfoxide in phosphate buffered salinewith 2.5% hexafluoroisopropanol were injected s.c. into separatetransgenic HLA A2.1 mice.

The methods referred to in Examples 9 and 10 were used to derive spleencell pollutions containing cells which were specific against pp65 andHCMV. The spleen cells from immunized mice were tested for recognitionand cytolysis against specific and non-specific peptide-sensitizedtargets, HCMV infected and non-infected HLA A*0201 and HLA-mismatchedfibroblast targets. The 495 pp65₄₉₅₋₅₀₃ peptide sensitized targets wereeffectively lysed, whereas targets sensitized with a peptide derivedfrom another protein (human p53 ) which binds to HLA A*0201 (p53₁₄₉₋₁₅₇)were ineffective. In addition, and importantly, HCMV-infected HLA A*0201fibroblasts were effectively lysed, but uninfected fibroblasts or thosewhich are HLA-mismatched were not recognized or lysed, regardless ofHCMV infection. These results were obtained with both molecules A and B,although molecule A was the more potent of the two. However, a secondimmunization (booster) of mice immunized with Vaccine A at lesseramounts of 50 nmole resulted in a detectable immune response when asingle immunization was not effective (Table 5). This stimulative effectof the booster is in agreement with B. Livingston et al., J. Immunol.159:1383-1392 (1997)). These data show that the single molecule form ofthe vaccine functions to stimulate HCMV immunity in an animal modelwithout any added molecules or adjuvant other than the vaccine moleculesA or B (Table 4) in liquid vehicle (PBS/DMSO/HFIP). The data alsodemonstrate that the vaccine is capable of stimulating a de novo immuneresponse to HCMV in a HCMV-naive mouse.

EXAMPLE 14

Additional HCMV pp65 CTL Epitopes Specific for HLA A and B Alleles

The experimental design used to determine the sequence of the 495peptide and its function is described in Examples 1-13. The 495 peptidespecifically bound to HLA A*0201 and the other subtypes shown in Example8.

There are over 100 known HLA Class I alleles of the A and B genes,however. (J. G. Bodmer et al., Tissue Antigens 49:297-321 (1997)). Usinga combination of predictive algorithms and truncation analysis,additional peptides from pp65 that will sensitize both autologous andallogeneic cells to be lysed by MHC-restricted human CTL wereidentified. As discussed in Example 1, the CTL arise from HCMVseropositive humans; therefore, the defined epitopes from pp65 werethose used by humans to suppress endogenous HCMV reactivation orviremia. To define CTL reactive against HCMV pp65 in combination withspecific HLA alleles, individual cloned CTL lines were tested forrecognition of EBVLCL infected with pp65vac which contained one of theHLA A or B alleles found in the individual whose blood was used toderive the CTL. In cases where at least one HLA allele is shared betweenthe EBVLCL targets and the CTL, sensitization for recognition and lysiswas observed. This experiment was repeated with at least threeindependent cell lines containing the restricting allele. Table 6 showsthe pp65 epitopes, their HLA restriction, the number of independent celllines of the same restriction which were sensitized, and the method(s)of delineation of the epitopes.

                  TABLE 6                                                         ______________________________________                                        HLA A or B                                                                              Number of                                                           Allele    Test Cell Method(s) of                                                                             Minimal Cytotoxic                              Specificity                                                                             Lines     Determination                                                                            Epitope Sequence                               ______________________________________                                        HLA A*0101 and                                                                          3         A, B, C, D, E                                                                            YSEHPTFTSQY                                    subtypes                       (SEQ ID NO:3)                                  HLA A*6801 and                                                                          3         A, B, D, E FVFPTKDVALR                                    subtypes                       (SEQ ID NO:5)                                  HLA B7 and                                                                              5         A, B, D, E TPRVTGGGAM                                     subtypes                       (SEQ ID NO:7)                                  HLA B*3502, 04,                                                                         3         A, B, C, D, E                                                                            FPTKDVAL                                       06 and subtypes                (SEQ ID NO:9)                                  ______________________________________                                         Methods of Determination: (A) HLA Restriction using single HLA allele         matched cell lines; (B) pp65 truncations in vaccinia viruses; (C) pp65        truncations in plasmids with detection of activity using TNFα assay     (D) matching of amino acid residues using published motifs; (E) functiona     analysis using ordered overlapping peptides.                             

EXAMPLE 15

Vaccine Molecules Comprised of More Than One CTL Epitope

In accordance with the procedures described herein, vaccines containinga combination of pp150 and/or pp65 epitopes, specific for the same ordifferent HLA Class I restriction elements, were prepared. Forvaccination of humans there is no necessity for each epitope to have thesame MHC restriction. A vaccine molecule which targets two or more MHCrestriction elements may be preferred because it allows the productionof fewer vaccine molecules, and still ensures that most HLA alleles weretargeted in a polymorphic pollution. Peptides with CTL epitopes whichare restricted by frequently expressed HLA alleles (see Tables 1 and 6)are preferred, and polypeptide vaccines containing epitopes from boththe pp65 and pp150 proteins, as well as an HTL epitope, are especiallypreferred for human vaccination against HCMV. The HLA alleles shown inTables 1 and 6 are a subset of possible HLA A, B and C CTL epitopes tobe potentially included in a multiple CTL epitope vaccine molecule. Theinclusion of multiple CTL epitopes and an HTL epitope will lengthen thepeptides (in the range of 40-50 amino acids). Therefore, thehydrophobicity of the sequence is an important factor. Alternatively,HCMV polypeptide vaccines may be prepared without a covalently attachedHTL epitope by using multiple CTL epitopes with a spacer of threealanine residues or another combination of amino acids between eachepitope, and two palmitic acid-lysyl amides at the N-terminus, as shownin Table 3.

EXAMPLE 16

Immunization of BMT Patients

A therapeutically active form of an antigenic peptide according to thepresent invention may be administered to an HCMV-seropositive bonemarrow transplant donor at a sufficient time (six to eight weeks, forexample) in single or multiple doses separated by a given number of daysor weeks prior to bone marrow transplant to enable the development of ananti-HCMV cellular immune response. The antigenic peptide can beformulated in per se known manner (for example, as a lipidated peptide,optionally in combination with a helper peptide and/or an adjuvant) andwill be administered, preferably, in multiple doses. If an unmanipulatedBMT graft will be given to the recipient, such a graft will contain 25%or more of mature T cells. The T cells will confer active immunity tothe BMT recipient patient. Alternatively, when a T-cell depleted BMTgraft is to be employed, an aliquot of T cells from the immunized donorcan be administered to the patient following (for example, approximately21 to 35 days) BMT in order to provide the recipient patient with HCMVimmunity.

EXAMPLE 17

In Vitro Assay for HCMV Infected Cells

The peptides of the present invention may be used in an in vitro assayto detect the presence or absence of HCMV-infected cells obtained, forexample, from a patient who's HCMV status (infected or uninfected) isunknown. T lymphocytes obtained from the patient are incubated with APCprimed with a peptide of the present invention. The activation of CTLsor CTLp's will reveal that the patient was infected with HCMV.

Although certain preferred embodiment and examples of the invention havebeen described, the invention is not so limited. Persons skilled in thisfield of science will understand that the present invention is capableof wide application in the fields of diagnostics and therapeutics, andthat modifications and variations can be made to the invention withoutdeparting from its spirit and scope.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 18                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Asn Leu Val Pro Met Val Ala Thr Val                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Domain                                                          (B) LOCATION: 2                                                     #/note= "Xaa = Leu, Ile, Met,ON:                                              #Val           Thr or                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Domain                                                          (B) LOCATION: 9                                                     #/note= "Xaa = Val, Ala, Cys,ON:                                              #or Thr"       Ile, Leu                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Asn Xaa Val Pro Met Val Ala Thr Xaa                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Tyr Ser Glu His Pro Thr Phe Thr Ser Gln Ty - #r                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Domain                                                          (B) LOCATION: 2                                                     #/note= "Xaa = Ser, Thr or Leu":                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Tyr Xaa Glu His Pro Thr Phe Thr Ser Gln Ty - #r                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Phe Val Phe Pro Thr Lys Asp Val Ala Leu Ar - #g                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Domain                                                          (B) LOCATION: 2                                                     #/note= "Xaa = Val or Thr"ATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Domain                                                          (B) LOCATION: 11                                                    #/note= "Xaa = Leu, Arg or Lys":                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Phe Xaa Phe Pro Thr Lys Asp Val Ala Leu Xa - #a                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Thr Pro Arg Val Thr Gly Gly Gly Ala Met                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Domain                                                          (B) LOCATION: 10                                                    #/note= "Xaa = Leu, Phe or Met":                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Thr Pro Arg Val Thr Gly Gly Gly Ala Xaa                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Phe Pro Thr Lys Asp Val Ala Leu                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Ile Leu Ala Arg Asn Leu Val Pro Met Val                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Glu Leu Glu Gly Val Trp Gln Pro Ala                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Arg Ile Phe Ala Glu Leu Glu Gly Val                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 29 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Lys Ser Ser Gln Tyr Ile Lys Ala Asn Ser Ly - #s Phe Ile Gly Ile Thr         #                15                                                           - Glu Ala Ala Ala Asn Leu Val Pro Met Val Al - #a Thr Val                     #            25                                                               - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 29 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Cys Ser Ser Gln Tyr Ile Lys Ala Asn Ser Ly - #s Phe Ile Gly Ile Thr         #                15                                                           - Glu Ala Ala Ala Asn Leu Val Pro Met Val Al - #a Thr Val                     #            25                                                               - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 28 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Domain                                                          (B) LOCATION: 6                                                     #/note= "Xaa =OTHER INFORMATION:                                                             cyclohexylal - #anine or Phe"                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Lys Ser Ser Ala Lys Xaa Val Ala Ala Trp Th - #r Leu Lys Ala Ala Ala         #                15                                                           - Gly Gly Gly Asn Leu Val Pro Met Val Ala Th - #r Val                         #            25                                                               - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 28 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Domain                                                          (B) LOCATION: 6                                                     #/note= "Xaa =OTHER INFORMATION:                                                             cylcohexylal - #anine or Phe"                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Cys Ser Ser Ala Lys Xaa Val Ala Ala Trp Th - #r Leu Lys Ala Ala Ala         #                15                                                           - Gly Gly Gly Asn Leu Val Pro Met Val Ala Th - #r Val                         #            25                                                               - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 25 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Domain                                                          (B) LOCATION: 6                                                     #/note= "Xaa =OTHER INFORMATION:                                                             cylcohexylal - #anine or Phe"                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Lys Ser Ser Ala Lys Xaa Val Ala Ala Trp Th - #r Leu Lys Ala Ala Ala         #                15                                                           - Asn Leu Val Pro Met Val Ala Thr Val                                         #            25                                                               - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 29 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                      -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Lys Ser Ser Ile Ser Gln Ala Val His Ala Al - #a His Ala Glu Ile Asn         #                15                                                           - Glu Ala Ala Ala Asn Leu Val Pro Met Val Al - #a Thr Val                     #            25                                                               __________________________________________________________________________

We claim:
 1. A peptide which elicits an MHC Class I cellular immuneresponse to human cytomegalovirus comprising a peptide selected from thegroup consisting of NX₁ VPMVATX₂, wherein X₁ is L, I, M, T, or V and X₂is V, A, C, I, L or T (SEQ ID NO: 2) with the proviso that the peptideis not NLVPMVATV (SEQ ID NO:1); YXEHPTFSQY, wherein X is S, T or L (SEQID NO: 4); FX₁ FPTKDVALX₂, wherein X₁ is V or T and X₂ is L, R, or K(SEQ ID NO: 6); TPRVTGGGAX, wherein X is L, M, or F (SEQ ID NO:8); andFPTKDVAL (SEQ ID NO: 9).
 2. A peptide according to claim 1, wherein thesequence of said peptide is NX₁ VPMVATX₂, wherein X₁ is L, I, M, T, or Vand X₂ is V, A, C, I, L or T (SEQ ID NO:2) with the proviso that thepeptide is not NLVPMVATV (SEQ ID NO:1).
 3. A peptide according to claim1, wherein the sequence of said peptide is YXEHPTFSQY, wherein X is S, Tor L (SEQ ID NO:4).
 4. A peptide according to claim 3, wherein thesequence of said peptide is YSEHPTFTSQY (SEQ ID NO: 3).
 5. A peptideaccording to claim 1, wherein the sequence of said peptide is FX₁FPTKDVALX₂, wherein X₁ is V or T and X₂ is L, R, or K (SEQ ID NO:6). 6.A peptide according to claim 5, wherein the sequence of said peptide isFVFPTKDVALR (SEQ ID NO: 5).
 7. A peptide according to claim 1, whereinthe sequence of said peptide is TPRVTGGGAX, wherein X is L, M, or F (SEQID NO:8).
 8. A peptide according to claim 7, wherein the sequence ofsaid peptide is TPRVTGGGAM (SEQ ID NO: 7).
 9. A peptide according toclaim 1, wherein the sequence of said peptide is FPTKDVAL (SEQ ID NO:9).
 10. A peptide according to claim 1, wherein said peptide islipidated.
 11. A peptide according to claim 1, wherein said peptide ismonolipidated.
 12. A peptide according to claim 1, wherein said peptideis dilipidated.